Cryo-electron microscopy (cryo-EM) is used to study the native, nanometer-scale 3-D structure of cells and cell organelles as well as the near-atomic-scale 3-D structure of biological macromolecules. While impressive advances in light microscopy enable detection and location of macromolecules in cells with nanometer-scale precision, fluorescence-based techniques detects only structures that are labeled. On the other hand, the technique of cryo-EM tomography reveals at once the 3-D interaction between all structural components of the cell. While x-ray crystallography can solve macromolecular structure at atomic resolution, the technique of "single-particle" cryo-EM can achieve near-atomic resolution without the need to crystallize the macromolecule;and while NMR can provide atomic resolution of small macromolecules in solution, cryo-EM can provide near- atomic resolution of macromolecules up to the mega-Dalton range. Cryo-EM depends on phase-contrast imaging of vitreously frozen specimens, which are weakly-scattering and very sensitive to electron irradiation. Thus it is critical to obtain the maximum contrast with the minimum electron dose. However, the currently employed method of phase-contrast imaging requires that the microscope be strongly defocused, which causes features in different size ranges to have different contrast. As a result, there is considerable overall loss of contrast, and complicated image-processing is needed when making a 3-D reconstruction. These shortcomings pose serious obstacles to increasing cellular resolution in cryo-EM tomography, to increasing image-processing throughput in single-particle reconstruction of macromolecules, and to extending single-particle reconstruction to macromolecules smaller than about 150 kDa,. The disadvantages of the defocus method of cyo-EM phase-contrast imaging can be overcome by in-focus imaging using a phase plate, as demonstrated in recent proof-of-principle studies. We propose to continue development of phase-plate imaging in order to make it a practical, routine technique for cryo-EM. We will (1) improve thin-film phase plate design and manufacture so that they can be widely and economically supplied and have adequate lifetimes, (2) adapt existing automated cryo-EM data-collection software for use with phase plates so that the phase plate stays centered and the optimum illumination conditions are maintained, and (3) establish protocols and guides to optimize phase-plate imaging for specific classes of specimens. This developmental work will have a major impact on the ability of cryo-EM to provide detailed knowledge about biological structures, both at cellular and molecular levels, and it will significantly increase throughput. PUBLIC HEALTH RELEVANCE: Nanomater-scale reconstruction of subcellular components in-situ in a native state, as well as near-atomic- resolution reconstruction of native macromolecules, depends on phase-contrast cryo-electron microscopy (cryo-EM). We propose to develop phase-plate imaging, a technique recently shown to overcome the limitations of conventional phase-contrast imaging, into a practical, routine method for cryo-EM studies. This will have a broad and major impact on biomedical research by expanding the applications and capabilities of cryo-EM.